Cardiovascular diseases which result from thrombosis of blood vessels are a leading cause of deaths. At present, the treatment is dissolution of the thrombus using a thrombolytic agent, namely a plasminogen activator (PA). Activation of plasminogen produces plasmin which degrades fibrin. Plasmin, however, also degrades clotting factors. Thrombolytic therapy which introduces systemic generation of plasmin, therefore, carries the risk of hemorrhage. Previously, we proposed a novel, pro-drug and triggered release approach which could permit targeted thrombolysis without the bleeding risk. The approach consists of two components: [i] a fibrin- targeting antibody linked to an anionic heparin (termed Ab-Hep); and [ii] a cation-modified PA (termed m-PA+). These two components are linked via an electrostatic interaction. Since the used cations are small, m-PA+ would retain its catalytic activity. This activity, however, would be inhibited after binding to Ab-Hep due to blockage of the PA's active site by the appended macromolecules. Since protamine is a clinical heparin- binding antidote, it can be used safely to trigger the release of m-PA+ from the Ab-Hep-m-PA+ complex. Thus, the approach would permit injection of a fibrin-targeting but inactive PA drug (thereby alleviating the bleeding risk by aborting systemic generation of plasmin), and subsequently a triggered release of the active m-PA+ in close proximity of a fibrin deposit. Although it was a brand new project with minimal data, the previous application received full support from NIH shortly after submission based on its scientific merits and clinical significance. For reasons not clearly stated, however, the overall grant period was cut by NIH from 4 to 3 years. Despite being handicapped by a shortened time for renewal, our group has made remarkable progress and outstanding productivity. In a short 2-year period, 20 manuscripts and 10 abstracts have been published or submitted. The in vitro feasibility of the project, particularly on the pro-drug and triggered-release features, have been demonstrated in plasma. In this new application, we plan to build upon those promising findings and further establish the project. Our integrated specific aims are: [i] develop analytical methods essential to the project; [ii] produce the desired m-PA+ by biological or chemical means; [iii] produce the Ab-Hep conjugates; [iv] test the functions of the final Ab-Hep-m-PA+ products in vitro; [v] examine their pharmacokinetic properties in rats; and [vi] test their functions in vivo using a rabbit jugular vein model and a clinically- simulated canine intracoronary thrombosis model.